Reactive oxygen species (ROS) can damage proteins, nucleic acids, and lipids. Methionine oxidation is one of the most common posttranslational modifications to proteins mediated by reactive oxygen species (ROS) that may alter protein structure/function. During conditions of oxidative stress, there is an accumulation of methionine sulfoxide (MetO)-containing proteins (MetO-proteins) (Moskovitz 1997, Moskovitz 1998). Insufficient reversal of MetO-proteins to those having unoxidized methionine by the methionine sulfoxide reductase system (Msr family consisting of MsrA and MsrB) may cause certain proteins to loose their function, aggregate, and be toxic to the cell (Moskovitz 1997; Moskovitz 1998; Gabbita 1999; Stadtman 2003). If not properly repaired, such damage may lead to the development of oxidative-stress related diseases. Alternation of methionine residues between their oxidized and reduced form could serve as a mean to control certain proteins function/activity. In addition, it has been suggested that methionines can function as ROS scavenging agents by their cyclic oxidation/reduction in the cell (Stadtman 2002).
The accumulation of damaged proteins (e.g. MetO-protein and protein-carbonyl) as a result of oxidative stress is well documented, especially in msrA null mutants of various species (Moskovitz 1997, Moskovitz 1998; Mostkoviz 2001). Oxidative damage to proteins is considered to be one of the major causes of aging and age-related diseases, and thus mechanisms have evolved to prevent or reverse these modifications. Pathology analysis performed on postmortem Alzheimer's diseased brains revealed higher levels of MetO-protein (Dong 2003) and carbonyl groups that correlated with diminished Msr activity in comparison to control brains (Gabbita 1999).
Identifying MetO targeted proteins will greatly enhance the knowledge about processes leading to cellular malfunction associated with protein damage, thereby providing information that could be pivotal in developing of novel therapeutics for treating oxidative stress-associated diseases. With regard to the effect of methionine oxidation on protein function, only a few specific proteins have identified as in vivo targets for such modification. The proteins include calmodulin (Gao 1998), Ikappa B (Kanayama 2002, Mohri 2002), and the voltage-dependent K (+) channel (Shaker) (Giorba 1997). The current knowledge about the identity of methionine-oxidized proteins, either in their fully damaged state or their intermediate functional stage in vivo, is still very limited.
One of the major reasons for this lack of extensive data is the fact that there is no direct and efficient screening method for the identification of cellular MetO-proteins. To date, specific proteins from biological extracts could be mainly monitored for their MetO moiety by following their purification, amino acid analysis and/or their analysis by mass spectrometry techniques. Several laboratories have attempted to develop antibodies specific to MetO in proteins. However, all such attempts failed, mainly due to the inability of developing an immunogenic MetO-containing antigen.
The present invention is directed to the production of antibodies specific for MetO-proteins. It is anticipated that the development of a new method for MetO-protein analysis will greatly advance research involving post-transnational modification to proteins. More specifically, identification of MetO-proteins in physiological processes that are affected by ROS production (e.g. aging and neurodegenerative diseases) will shed light on cellular processes that can become toxic due to the accumulation of specific methionine-oxidized proteins.